The invention relates to the field of amplification and sequencing of polynucleotides and/or proteins as well as fragments of such compounds. More particularly, it relates to methods for analyzing their structure and molecular weights as well as monitoring the progress of processes wherein such compounds are made.
There is an increasing demand for reliable and inexpensive methods for the sequencing of polynucleotides, such as, DNA, RNA and the like. Generally, a radioactive or fluorescent probe, which selectively hybridizes to a specific target nucleic acid, is added to the support. A typical common type of probe is a single-stranded (ss) DNA which is complementary to a sequence in the target DNA or RNA.
The hybrid molecule thus formed with the label probe thereon may then be detected by various techniques depending on the nature of the label used. An example of such hybridization is shown in U.S. Pat. No. 4,358,535.
Typical labelling probes include the incorporation of a radioactive atom, such as, .sup.32 P, .sup.14 C, or .sup.3 H. This can be achieved by nick translation, such as that shown in Rigbny et al (J. Mol. Biol., 113:237, 1977), wherein a labelled nucleotide is incorporated into a gap created in the DNA of the probe. Other labels can be introduced by nick translation, for example, by incorporating biotinylated nucleosides which can then be coupled to an avidin bound label, such as, an enzyme. The DNA can also be labelled with antigenic groups reacting with antibodies.
For the assay or quantification of nucleic acids, such as, DNA or mRNA, either the total nucleic acid material present in the sample or that transcribed from a specific gene can be conventionally determined by this so-called dot-blot analysis technique.
One of the problems with such sequencing techniques requires the handling of radioactive isotopes and presents an environmentally undesirable situation in the laboratory. The use of fluorescent labels or enzyme labels results in relatively complicated techniques for the ultimate reading of the label. Generally, expensive equipment and relatively skilled technicians are needed to effect the analysis of gels and/or substrates onto which the labelled molecules or segments have been fixed.
In addition, numerous attempts have been made to automize the electrophoresis step, detection and data handling. However, difficulties have arisen with respect to such methods primarily because of the method of labelling.
The present invention also relates to methods for the amplification, i.e., duplication of polynucleotides, such as, DNA. Numerous methods for duplication have arisen. These include the so-called polymerase chain reaction (PCR) which is based on the phenomenon known as primer extension by DNA polymerases. In this method, an oligonucleotide having a size of 15 to 20 base-pairs is used as a primer which is complementary to the 5' end of each strand of a sequence tagged site. The primer is mixed in excess with a DNA sample from a target sequence (STS) to be amplified. The 4-deoxyribonucleoside triphosphates are also provided. The reaction mixture is then taken through a sequence of multiple synthetic cycles which consist of a first denaturization to take apart the two strands of DNA and create a set of single stranded templates, annealing by cooling to encourage the primers to anneal to their complementary sequences on a single stranded templates, and then heating to activate the polymerase which results in extension of a new DNA strand.
The reaction mixture is normally taken through a multiple of such synthesis cycles depending on how much amplification is desired. It is desirable to be able to determine at some point during the process the status of the synthesis, i.e., how much new DNA has been produced. To date, the methods provided for such determination have generally not proven satisfactory.